Long Noncoding RNA (lncRNA) Revisited

  • Publications Dealing with lncRNAs Show Exponential Growth
  • Evidence for Involvement of lncRNAs in Cancer is Increasing
  • Value of lncRNAs as Biomarkers Has Been Validated

Several years ago, I posted a blog about long noncoding RNAs (lncRNAs), which are defined as non-protein coding transcripts in the range of ~200 nt to ~100 kb long. Interest in lncRNA—and other types of noncoding RNA such as microRNA (miRNA) and short interfering RNA (siRNA)—is fueled in large part by a collective scientific desire to uncover and understand the existence and function of all forms of RNA dark matter, so named by analogy to dark energy in cosmology. The lncRNA component of RNA dark matter is certainly generated from transcription of noncoding (formerly “junk”) DNA, but much has yet to be elucidated about function.

As depicted below, lncRNA (red) may act as (a) decoys to release proteins from chromatin, (b) scaffolds for grouping protein complexes, (c) guides to recruit proteins or (d) transcriptional enhancers by bending chromatin. Not shown is lncRNA acting as an antagonist for other regulatory noncoding RNAs, namely miRNA, which can be studied by next-generation sequencing methods such as TriLink’s CleanTag approach.

Taken from Bohla et al. Dis Markers (2017)

Numerical support for upward trending interest in lncRNA is provided by the chart shown here for the number of annual publications on lncRNA. This chart was produced by using data I found in PubMed for 2000-2016, which clearly show a relatively flat rate of ~150 papers per year from 2000-2007, and then an exponential increase to ~2,220 papers in 2016.

Although it’s not possible to say for sure what catalyzed this marked upturn in lncRNA publications, searching the 2005 literature in Google Scholar led to finding the following top-5 cited publications, which from titles alone could be likely scientific co-catalysts:

In any case, when I did keyword searches of the ~13,000 publications on lncRNA, roughly one-third (~4,700) were related to cancer, and many (~1,800) dealt with biomarkers primarily for (~1,400) cancer, but also including cardiovascular diseases, diabetes, epilepsy, general anxiety disorder, inflammatory bowel diseases, etc. Given that cancer is a major medical problem for all countries to deal with, and knowing that early detection of cancer by finding better biomarkers is critically important, this blog revisits lncRNA in the context of cancer and biomarkers.

State-of-the-Art Technologies to Explore lncRNA

At the risk of over simplification, advances in RNA sequencing (RNA-Seq)—enabled largely by high-throughput instrumentation from Illumina, PacBio, and Thermo Scientific—has revolutionized the field of molecular biology by revealing that up to ~75% of the human genome is actively transcribed, and that most of this transcriptome consists of lncRNA. Bioinformatic analyses, which are way beyond my expertise, have played a key role in sorting out lncRNA from mRNA and other RNA species. Interested readers can consult Cobos et al. as a recent lead reference to learn more about these bioinformatic methods.

Following are links to a constellation of additional experimental techniques that are available for exploring lncRN, and can be perused in detail later:

Although these methods are employed to shed light on lncRNA cellular localization, structure, interaction networks and functions, interested readers should consult a review by Salehi et al. and research paper by Goyal et al. for discussions of the advantages and disadvantages of these techniques. For example, Goyal et al. note that many lncRNA are derived from bidirectional promoters, or overlap with promoters, or bodies of sense or antisense genes. In a genome-wide analysis, they found only 38% of 15,929 lncRNA loci are safely amenable to CRISPR applications, while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. For several representative lncRNAs, it was found that CRISPR—but not RNAi by siRNA or antisense oligos—also affects their respective neighboring genes.

In closing this section on methods, readers who follow my blogs know that I’m a big fan of nanopore sequencing, about which I’ve commented in several previous posts. Oxford Nanopore Technologies has recently announced advances in its nanopore technologies that now allow sequencing of an RNA strand directly, rather than analyzing the products of reverse transcription and PCR reactions. My scientific “crystal ball” sees use of nanopore sequencing of lncRNAs in the not too distant future.

Long Noncoding RNA in Cancer and as Biomarkers

According to Bohla et al., lncRNAs are now known to function as regulatory factors for numerous, important cellular processes, such as growth, differentiation, and cell death. In addition, lncRNAs are involved in controlling alternative splicing, regulation of gene expression at the posttranscriptional level, chromatin modification, inflammatory pathologies, and—when deregulated—various types of cancer. Lnc2Cancer is a manually curated, interactive database of cancer-associated lncRNAs with experimental support that provides a high-quality and integrated resource for exploring lncRNA deregulation in various human cancers. In my opinion, Lnc2Cancer is definitely worth perusing.

The figure shown here indicates various types of cancers (black) for which lncRNAs (names of which are in green, blue or red) have been implicated. Searching PubMed or Google Scholar using any of the lncRNA names will provide a host of publications to peruse.

Taken from Vitiello et al. Cellular Oncology (2015)

For example, searching PubMed for the term “HOTAIR” gives ~475 publications, in chronological order starting with the most recent. By contrast, searching Google Scholar for the terms “HOTAIR lncRNA” gives ~6,100 articles ranked by “relevance,” which is explained elsewhere as being heavily influenced by citation frequency. Readers interested in more details can consult Lin & Yang, who review the mechanisms by which lncRNAs regulate cellular responses to extracellular signals, and discuss the clinical potential of lncRNAs as diagnostic indicators, stratification markers and therapeutic targets of combinatorial treatments.

As was mentioned in the introduction, there are numerous publications aimed at using these and other cancer-associated lncRNAs as biomarkers. Among the main advantages of lncRNAs that make them suitable as cancer diagnostic and prognostic biomarkers, high stability while circulating in body fluids, especially when included in exosomes or apoptotic bodies, is noted by Bohla et al. Despite abundant quantities of ribonucleases in different body fluids, lncRNAs protected in exosomes or apoptotic bodies can be detected in whole blood, plasma, urine, saliva and gastric juice. These lncRNAs as biomarkers are obtainable by non- or minimally invasive methods, which are well tolerated by patients compared to conventional biopsies.

Taken from liquid-biopsy.gene-quantification.info

Challenges for use of lncRNAs as biomarkers include development of convenient, low cost yet robust isolation methods, and accurate quantitation of relatively low copy numbers, which heretofore has relied on an amplification step, such as enzymatic conversion into cDNA followed by PCR. However, single-molecule detection approaches have evolved to obviate the need for amplification.

For example, NanoString Technologies now offers the nCounter® lncRNA Assay for validation of lncRNA discoveries, which can then be followed by use for biomarker quantification. As depicted below in the left panel, single molecules of lncRNAs (red) can be detected at the same time as mRNAs green and blue), if so desired, using sequence-specific probes each having a fluorescence-based “barcode” identifier. The right panel depicts extension of this approach to identify lncRNA-protein interactions by inclusion of antibody precipitation. This digital-counting assay allows researchers to select up to 800 lncRNAs for analysis in a single multiplexed reaction, which is quite impressive, in my opinion.

Taken from nanostringxt.com

Taken from Cesano J Immunother Cancer (2015)

Readers interested in more details for application of nCounter® for analysis of biomarkers are referred to a recent publication by Permuth et al. dealing with pancreatic ductal adenocarcinoma (PDAC), which is an aggressive disease that lacks effective biomarkers for early detection. Briefly, these researchers hypothesized that circulating lncRNAs may act as diagnostic markers, and used nCounter® technology to measure the abundance of 28 candidate lncRNAs in pre-operative plasma from a cohort of pathologically-confirmed PDAC cases of various grades of severity and non-diseased controls. Results showed that two lncRNAs aided in differentiating PDAC from controls, and an 8-lncRNA signature had greater accuracy than standard clinical and radiologic features in distinguishing ‘aggressive/malignant’ PDAC that warrant surgical removal from ‘indolent/benign’ PDAC. In my opinion, these findings seem very promising for use with PDAC and, by conceptual extension, to other cancers.

Temozolomide (TMZ). Taken from sigmaaldrich.com

As a final example of lncRNA biomarkers for cancer, MALAT1 (pictured above) has been recently reported by Chen et al. as a prognostic factor in glioblastoma multiforme (GMF), and induces chemoresistance to temozolomide (TMZ). The significance of these findings is that GBM is the most malignant brain tumor with limited therapeutic options, and that TMZ is first-line chemotherapy for GBM. These researchers first used deep-sequencing and bioinformatic methods to identify lncRNAs showing different expression levels in TMZ-resistant and non-resistant patients. RT-qPCR was then performed in tissues and serum samples, and lncRNA MALAT1 was shown to discriminate between responding patients from non-responding patients.

Closing Comments

If you’re are interested knowing much more about lncRNAs, then lncRNABlog.com is a great website for you to visit and subscribe to, if you want to keep current on all manner of lncRNA research and industry news. This interactive blog, which posts abstracts and images from the latest lncRNA publications, allows readers to post comments that allow you to join in the “conversation” or simply follow what others are thinking about these articles.

Also provided are links to a host of different online tools for lncRNA research and development, as well as “what’s happening” in terms of upcoming lncRNA events or conferences. Those of you currently seeking a new position may find the jobs postings to be helpful.

Finally, there are there plenty of pop-up advertisements and commercial banners, but these are also informative about lncRNA products and services that are available.

As usual, your comments are welcomed.

Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies

Backstory

Taken from sysmex-inostics.com 

Taken from sysmex-inostics.com

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.

CTCs

In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from journal.frontiresin.org

Taken from journal.frontiresin.org

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from nature.com

Taken from nature.com

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from nature.com

Taken from nature.com

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from miroculus.com

Taken from miroculus.com

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from graymatters.com

Taken from graymatters.com

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Small RNA is Big Science

  • Most Top-5 Citations in Clinical Chemistry are MicroRNA (miRNA) Biomarkers
  • miRNA Biomarker Bonanza is Predicted by Panel of Experts, Although No miRNA Biomarkers Have Yet Been Approved by FDA
  • Plethora of Potential Short Regulatory RNA Exists Beyond the Typical miRNA Microcosm

I’m always looking for new and hopefully engaging topics to comment on, and a recent “Best of Clinical Chemistry” item featured in a special issue of Clinical Chemistry definitely caught my attention. I wasn’t surprised by MIQE Guidelines being at the top, given that these are the “bible” for doing accurate quantitative PCR (qPCR) that has become a seemingly ubiquitous molecular assay for clinical studies. However, I was totally surprised that the next four “best of” all involved microRNA (miRNA)! Hence today’s blog about these small RNA being big science—play on words intended (although properly speaking I should say short rather than small). Continue reading

Pseudouridine Biomarker for Breast Cancer

As you are probably aware, October is National Breast Cancer Awareness month. Everyone from the NFL to Yoplait yogurt seems to be engaged in campaigns for fundraising and awareness. I think it’s great to see the extensive community support for this worthy cause. Since breast cancer awareness is top of mind this month, I thought I’d follow up my latest blog about pseurdouridine with a ‘mini blog’ highlighting some interesting research involving a pseudouridine biomarker for breast cancer.

I’d also like to mention that TriLink is participating in Breast Cancer Awareness month. For every order placed in October, TriLink will dontate $5 to Susan G Komen to support the upcoming 3-Day Walk being held November 20-22 in San Diego.

3day

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Gene-Expression Biomarkers Can Detect Depression

  • First-ever Lab Test for Depression Found Using RT-PCR
  • FDA Approval as Diagnostic Possible by Early 2016
  • Huge Potential Market as 1-in-10 US Adults Suffer from Depression

While it’s normal for everyone to occasionally feel blue or sad, prolonged bouts of depression that interfere with normal life are indicative of a serious mental health issue. While there are numerous forms and differing severity of depressive disorders, as described at a National Institute of Mental Health (NIMH) website, only two factual aspects of this illness really stand out in my opinion:

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

  • Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.
  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.

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Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics

  • Invasive Needles and Scalpels Seen as Passé
  • Noninvasive Sampling Advocates Focusing on Circulating Tumor Cells (CTCs) 
  • New Companies are Pursuing the Liquid Biopsy “Gold Rush”

Biopsy Basics

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

As defined in Wikipedia, a biopsy is ‘a medical test commonly performed by a surgeon or an interventional radiologist involving sampling of cells or tissues for examination.’ Biopsies can be excisional (removal of a lump or area), incisional (removal of only a sample of tissue), or a needle aspiration (tissue or fluid removal). Despite the value of these traditional types of biopsies, they are more or less invasive, lack applicability in certain instances, and require accurately “going to the source” of concern, as pictured to the right, for ultrasound-guided breast cancer biopsy. Better methodology is highly desirable and is the topic of this post. By the way, if you want to peruse a lengthy list of scary risks associated with various type of common invasive biopsies, click here to see what I found in Google Scholar by searching “incidence of complications from biopsies.”

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